ABSTRACT

Introduction: Oral corticosteroids are the cornerstone of acute asthma management in the emergency department. Recent evidence has raised doubts about the efficacy of this treatment in preschool-aged children with viral-induced wheezing and in smoking adults. The aims of the study were to: (1) document the magnitude of response to oral corticosteroids in children presenting to the emergency department with moderate or severe asthma; (2) quantify potential determinants of response to corticosteroids and (3) explore the role of gene polymorphisms associated with the responsiveness to corticosteroids.

Methods and analysis: The design is a prospective cohort study of 1008 children aged 1–17 years meeting a strict definition of asthma and presenting with a clinical score of ≥4 on the validated Pediatric

Respiratory Assessment Measure. All children will receive standardised severity-specific treatment with prednisone/prednisolone and cointerventions (salbutamol with/without ipratropium bromide). Determinants, namely viral aetiology, environmental tobacco smoke and single nucleotide polymorphism, will be objectively documented. The primary efficacy endpoint is the failure of emergency department (ED) management within 72 h of the ED visit.

Secondary endpoints include other measures of asthma severity and time to recovery within 7 days of the index visit. The study has 80% power for detecting a risk difference of 7.5% associated with each determinant from a baseline risk of 21%, at an α of 0.05.

Ethics and dissemination: Ethical approval has been obtained from all participating institutions. An impaired response to systemic steroids in certain subgroups will challenge the current standard of practice and call for the immediate search for better approaches. A potential host–environment interaction will broaden our understanding of corticosteroid responsiveness in children. Documentation of similar effectiveness of corticosteroids across determinants will provide the needed reassurance regarding current treatment recommendations.

Results: Results will be disseminated at international conferences and manuscripts targeted at emergency physicians, paediatricians, geneticists and respirologists.

Trial registration number: This study is registered at Clinicaltrials.gov (NCT02013076).

INTRODUCTION

Asthma is the most common chronic disease in childhood. It affects over half a million Canadian children aged 4-11 years. Of all respiratory illnesses, asthma is one of the most frequent paediatric diagnoses requiring hospital admission. The burden of illness is much higher in preschool-aged children, who account for over 50% of emergency department (ED) visits and who have three times the hospital admission rate of older children and adolescents. The difficulty in measuring asthma severity in young children has resulted in a dearth of comparative therapeutic evidence between preschoolers and older children.

Treatment of acute asthma

National and international guidelines for the manage- ment of acute asthma recommend: (1) inhaled β2-agonists for all patients; (2) systemic (usually oral) corticosteroids for those with moderate and severe asthma; and (3) repeated doses of inhaled β2-agonists and anticholinergics for severe cases. The latter two recommendations independently reduce admission rates by 25% in studies of predominantly school-aged children. Several features must be noted. First, recommendations are severity- specific; patients with mild asthma do not appear to benefit from oral corticosteroids. Second, the concept of the ‘golden first hour of treatment’ supports early and aggressive asthma management, as the risk of admission is reduced only when oral corticosteroids are administered 3–4 h prior to the decision to admit. Third, of all treatments administered in the ED, oral corticosteroids are by far the most effective for preventing admission. Fourth, there is no equally effective substitute for oral corticosteroids; promising contenders such as high-dose inhaled ster- oids, intravenous antileukotrienes or magnesium sulfate have been shown to be inferior to oral corticosteroids and are used as an add-on therapy. Fifth, systemic corticosteroids are inexpensive generic drugs. Short treatment with oral corticosteroids is generally devoid of significant side effects, though rare cases of fatal or disse- minated varicella have been reported. Sixth, while recommendations are relatively similar in young children, older children and adults, the evidence for the former is weaker due to the under-representation of preschool-aged children in relevant trials. While oral corticosteroids are the cornerstone of management of acute, moderate or severe asthma, several reports have recently shaken the belief that they are equally effective for all patients with asthma, showing that children with viral-induced wheezing and smoking adults are corticosteroid-resistant. Indeed, in a large placebo-controlled randomised con- trolled trial of 700 children aged 10–60 months with mild-to-moderate viral-induced wheezing, oral corticosteroid was not superior to placebo for reducing the length of stay in hospital or improving the Pediatric Respiratory Assessment Measure (PRAM) clinical score, despite adequate study power. Critics have suggested that: (1) in most children, the disease may not have been asthma (documented in only 16% of patients) but rather the North American definition of bronchiolitis; (2) some patients (with mild asthma) may not have needed corticosteroids; (3) the prolonged stay in hospital did not seem to be supported by severity and (4) the dose of 1 (instead of 2) mg/kg may have been insufficient. Yet, this study elicited major discomfort regarding acute asthma management in young children. In another adequately powered landmark trial, a 2-week treatment with prednisone showed a marked blunting of response in adult smokers, with an improvement in forced expiratory volumein1s(FEV1)of237mL(95%CI43to431)in never-smokers compared to no change or 47 (−148 to 243) mL in current smokers. The question remains as to whether smoking adolescents and children with environ- mental tobacco smoke (ETS) respond as well as those not exposed. None of these aforementioned findings have been replicated, thus prompting us to review the potential determinants of responsiveness.

Potential determinants of response

Disease diagnosis

Children with bronchiolitis do not respond to oral corti- costeroids, although a recent trial has demonstrated a sig- nificant response only in association with nebulised epinephrine. It is thus critical to distinguish asthma from bronchiolitis in young children. Bronchiolitis is clinically defined in North America as the first wheezing illness in a child ≤12 months, with respiratory syncytial virus (RSV) as the most frequent pathogen. Asthma, defined as airway obstruction (cough, dyspnoea and wheezing) with hyper-reactivity and reversibility with bron- chodilator or corticosteroids, begins early in life. While children and adults meeting these criteria can be diagnosed at the first episode, generally three wheezing episodes are required for children ≤12 months to reduce the risk of misclassification with bronchiolitis. Heterogeneous groups of children with bronchiolitis and asthma may explain the poor response to oral corticoster- oids in studies including infants and toddlers. Thus, an operational definition of asthma, clinically applicable to children aged 1–17 years, is needed to reasonably exclude bronchiolitis.

Upper respiratory tract infections

Upper respiratory tract infections (URTIs), usually viral in origin, are the most frequent (60–80%) triggers of asthma exacerbation in children. RSV, parainfluenza virus and rhinovirus are frequently implicated in children under 2 years of age, while picornavirus, corona- virus and influenza are usually associated with asthma in older children. The incidence of viral-induced asthma peaks in September continues throughout the fall and winter. In adults with acute asthma, viral infection is associated with longer hospital admission and increased sputum neutrophils, suggesting a predominantly neutrophilic airway inflammation. In a study of children aged 3–36 months, those infected with rhino- virus showed fewer relapses when treated with oral prednisone compared to placebo, suggesting that rhinovirus did not impair responsiveness to steroids. In another placebo-controlled trial of 283 young children with wheezing, prednisolone did not significantly decrease the overall time to discharge; however, it reduced by half the length of stay in children infected with picornavirus and by fourfold that of children with enterovirus, sug- gesting that response may be organism dependent. Clearly, oral corticosteroids may not be as effective in patients with viral infections as in those without, perhaps due to neutrophilic airway inflammation, a condition associated with poor response to corticosteroids. Moreover, response may be organism specific, a hypothesis that requires careful documentation of aetiology.

Exposure to tobacco smoke

In addition to the aforementioned trial demonstrating no response to oral corticosteroids in asthmatic adult smokers, a blunted response to inhaled corticosteroids was also documented in adult smokers in two randomised controlled trials. While the mechanism behind the lack of response is not known, one can cer- tainly point to smoking’s direct toxicity, proinflammatory action or interference with the transcription of genes associated with corticosteroid response. Indeed, smoking has frequently been associated with airway neu- trophilia. In paediatrics, exposure to tobacco smoke has been associated with a higher incidence of URTIs and prevalence of asthma, and a greater severity of exacerbations. However, the impact on the therapeutic response has not been documented in children, as asthma trials have not examined or failed to report sub- group analyses on ETS exposure or active smoking. Heavier ETS exposure in preschoolers who spend more time at home than school-aged children may explain a poorer response in young children. Objective documentation of smoking would also be important if possible, even before high school where many already smoke. Of particular concern is whether a blunted response to oral corticosteroids would be found in chil- dren exposed to tobacco smoke and adolescents with a short history of active smoking. With 25% of asthmatic children exposed to ETS and 25% of teenagers actively smoking, such an assessment appears critical.

Several reports highlight the need for objective measure- ment of nicotine exposure, because of parental under- reporting of their child’s exposure to ETS. Cotinine, a nicotine metabolite with a half-life of 20 h, is a widely accepted indicator of recent tobacco use and exposure. Serum cotinine requires blood sampling, a major deter- rent for study participation in paediatrics. Good corre- lations exist between serum and either salivary (r=0.71) or urine (r=0.69) cotinine measures. With a 1:10 ratio of cotinine level between saliva and urine, passive exposure may lead to saliva levels of 5–10 ng/mL and urine levels of 10–100 ng/mL. However, as urine sampling in young children may not occur for several hours and is more complex to obtain in children not yet toilet-trained, saliva cotinine measured by the quantitative enzyme immunoassay kit is a well-validated, non-invasive solution that offers required precision with minimal volume and a detectable level of 0.05 ng/mL.

Other determinants

A number of other factors could possibly modulate the responsiveness to oral corticosteroids including, among others, gender, race, perceived asthma phenotype (derived from age, common asthma triggers and interim symptoms), inhaled corticosteroids, allergens, the alleged trigger, and other environmental triggers, all of which can be documented clinically by questionnaire.

Mechanistic pathways

Two promising mechanistic pathways may explain the variations in the magnitude of response observed in clinical effectiveness, namely (1) gene polymorphisms that may reveal potential gene–environment interactions and (2) the type of airway inflammation.

Gene polymorphisms

There is increasing evidence that inherited genes are not a deterministic genotype, but rather a genotype that encodes a potential range of phenotypes that will develop in response to a variety of environments. Consequently, a minor polymorphism between individuals in genes that modulate response to corticosteroids may predispose some people to environmentally induced problems, such as smoke-induced or viral-induced asthma. Two major groups of genes are of interest: (1) those affecting suscepti- bility to asthma and (2) those directly interfering with response to corticosteroids by coding for major compo- nents of the pathway involved in corticosteroid action (table 1). In the first group, we selected eight polymorph- isms in seven genes that can be divided into those coding for xenobiotic metabolising enzymes and those coding for mediators of inflammation and immunity, specifically the ones demonstrated to affect lung function, disease severity and interaction with exposure to ETS. We selected polymorphisms affecting gene function, top-ranking single-nucleotide polymorphisms (SNPs) in a number of associated studies. Transforming growth factor β 1 poly- morphisms were found to correlate with disease severity; CC16 polymorphisms play a role in the development and persistence of the asthma phenotype in childhood; CD14 polymorphisms have been linked to pathogenesis of asthma and lung function in smokers. The ORMDL3 gene confers susceptibility to early-onset asthma, particu- larly through interaction with early life exposure to ETS; GSTM1 and GSTP1 are involved in the metabol- ism of polycyclic aromatic hydrocarbon derivatives and reactive oxygen species; GSTM1 null and GSTP1 geno- types have been associated with an increased risk of asthma and rapid decline of lung function among smokers. The ADRB2 receptor gene was found to con- tribute to the occurrence of wheeze among children who were exposed to tobacco smoke in utero and in early child- hood and was related to β2-agonist response. Among genes affecting the corticosteroid and inflammatory

pathways, we selected genes (CHRH1, TBX21, FCER2, GLCC1, STIP1 and SERPINE1) that were shown to correlate with response to corticosteroids in patients with asthma; the finding for some of them was replicated in several cohorts. All selected polymorphisms with the gene name, position and corresponding reference are summarised in table 1. Genotyping is essential to link the observed corticosteroid response to genotype and to evalu- ate the potential host–environment interaction.

Airway inflammation

Induced sputum is a valid, reproducible and non-invasive method of assessing the magnitude and pattern of airway inflammation in adults and children. Sputum cell counts and differentials (ie, eosinophils and neutrophils) deter- mine the inflammatory phenotype; analysis of supernatants for eosinophil cationic protein (ECP), interleukin-8 (IL-8) and myeloperoxidase provides measures of cellular activa- tion. Sputum eosinophils and ECP increase with exposure to allergens and decrease with corticosteroid treatment. A higher proportion of neutrophils is associated with smoking and with viral infection. The non-eosinophilic inflammatory phenotype in adults has been associated with poor response to corticosteroids. Markers of this phenotype include an increased proportion of neutrophils, IL-8 and myeloperoxidase, all of which are easily quantifiable. Contrary to adult findings and cri- teria, three distinct inflammatory cell patterns have been reported during paediatric exacerbations: non-eosinophilic (54% neutrophils) in 35%, with paucigranular inflammation not described in acute paediatric asthma. Combined eosinophilic/neutrophilic exacerbations show more mast cells and higher sputum ECP levels than eosinophilic exacerbations. There is increasing evidence that eosinophilic asthma is more responsive to corticosteroids than non-eosinophilic asthma; a promising mechanistic pathway to explore.

Expired nitric oxide (eNO) is increasingly recognised as a non-invasive marker of airway inflammation, particularly in atopic asthma, and correlates strongly with the per- centage of airway eosinophils (r=0.78) and ECP (r=0.53) in children with asthma. Although the understanding about the link between NO and airway inflammation remains incomplete, airway epithelial cells activated by inflammatory cytokines produce an increased amount of eNO due to the expression of inducible NO synthase. eNO is elevated in untreated asthma and improves with asthma therapy. Of interest, eNO correlates with other markers of eosinophilic inflammation, including sputum eosinophils. Thus, eNO appears to reflect the magnitude of eosinophilic airway inflammatory and may serve as a promising biomarker of eosinophilic inflam- mation in children too young or unable to cooperate with induced sputum sampling. As it can be measured in preschool-aged using an offline technique and in older children using commercially available instru- ments, it could serve as a biomarker of subsequent response to corticosteroids if indeed the type of airway inflammation modulates response.

Selection of outcomes

Healthcare service utilisation

The need for hospital admission is a powerful marker of therapy failure, likely to alter physicians’ practice and influence decision makers as hospital costs alone account for 43% of total asthma costs. Although subject to prac- tice variation, a physician’s decision to admit is usually based on an unsatisfactory response to bronchodilators and systemic corticosteroids in the ED, indicating severe asthma or poor response to corticosteroids. Admission may be affected by other reasons such as parental anxiety, distance or fatigue and, of course, availability of ED and hospital beds. Adding a cut-off length of ED stay, above which a patient is considered admitted, limits the impact of external factors (bed availabilities) which may vary widely within and between institutions. Moreover, incorp- orating return visits resulting in admission provides an additional measure of failure of ED treatment by adding a measure of the decision appropriateness. Time to meet severity criteria for discharge (PRAM

Indicators of airway dysfunction

Accurate and objective assessment of the severity of airway obstruction is clearly the biggest challenge in acute paedi- atric asthma research. Standard lung function tests, such as spirometry, require a forced expiratory manoeuvre that is extremely difficult and unreliable for preschool-aged children because of poor coordination; it is not obtainable in 35–50% of acutely ill school-aged children because of illness severity and/or poor familiarity with technique. About three quarters of asthmatic children cannot perform standard lung function tests in the ED setting. In contrast, respiratory resistance (Rrs) by forced oscilla- tion is an effort-independent measure that can be obtained in untrained, acutely ill children aged ≥3 years We and others have demonstrated the reproducibility and sensitivity to change Rrs and established reference values for Canadian children. Thus, the measure of Rrs can serve as a precise and reliable index of severity and response in acutely ill children, although still missing very young or uncooperative preschoolers and as such cannot serve as the main outcome.

Clinical scores appear to be a reasonable alternative to lung function testing as they can be used in children of all ages. Although many clinical scores have been designed, only two have been validated for use in both preschool-aged and school-aged children. The PRAM is the only score developed and validated against a concurrent measure of lung function in children aged 3–6 years, and subsequently validated in children aged 1–17 years. It is a discriminative and responsive tool, with a change in scores ≥3 indicating clinical importance. The inter-rater reliability is consistently above 70%. The 12-point PRAM, rating five weighted items (oxygen satur- ation, suprasternal retractions, scalene muscle contraction, air entry and wheezing), has been used in landmark clin- ical trials. It can be used to assess severity at baseline (mild: PRAM 0–3; moderate: PRAM 4–7; severe: PRAM 8–12), improvement with treatment depicted as the area under the curve, need for admission (PRAM ≥4, 4 h after the oral corticosteroids) and time until ready for discharge (delay until PRAM

Indicators of recovery following discharge

Among children discharged home, the time to complete recovery will vary widely between individuals. Although symptom scores are frequently used in clinical trials, only four have been specifically developed for use in chil- dren. The Asthma flare-up diary for young children (formerly the Pediatric Asthma Diary) is the only one vali- dated to detect day-to-day change in the functional status of preschool-aged children following an acute care visit118; it is highly sensitive to detect group differences in intensity and duration of symptoms in randomised controlled trials; however, its performance in school-aged children has not been explored. Another diary has been validated for detecting change in school-aged children but not in the context of acutely ill children. Use of β2-agonists is an additional marker for the duration and intensity of symp- toms, as greater use of rescue relievers is expected to occur in children with more symptoms. Finally, the impact of the disease on the quality of life of the patient or the caregivers is a recognised, sensitive and unique marker of the burden of disease on the patient or family. Several well-validated asthma-specific quality of life instru- ments are available for adults and school-aged children with stable asthma. To our knowledge, only one instru- ment, entitled ‘Effects of a child’s asthma flare-up on parents’, is available for measuring the burden of disease on parents of preschool-aged children following an asthma exacerbation; we have developed the instrument using Kirshner and Guyatt’s approach for developing health instruments, validated it in a randomised controlled trial and received two copyrights (French version: #1019528 English version: #1019529 (manuscript in preparation).

Hypothesis

In children presenting with moderate or severe asthma to the ED: Preschool age, specific viral triggers and exposure to tobacco smoke are independently associated with increased risk of failure of ED management (risk dif- ference (RD) ≥7.5%). Specific genetic polymorphisms alone or in associ- ation with environmental factors are associated with higher risk of admission. An eosinophilic pattern of airway inflammation (ie, percentage of eosinophils on induced sputum and/ or baseline eNO) is associated with greater clinical improvement as measured by the PRAM area under the curve over 4 h (nested cohort).

OBJECTIVES

The main objective was to identify and quantify clinically available factors (eg, age, specific viral trigger and ETS) associated with failure of ED management and other markers of clinical response to oral corticosteroids in preschool-aged and school-aged children presenting to the ED with moderate or severe asthma.

Secondary objectives

Determine whether the type of airway inflammation (ie, eosinophilic and eNO) is associated with the magnitude of response to corticosteroids and which host or episode characteristics are associated with a specific type of inflammation.

METHODS

Design

The study design is a multicentre prospective cohort study with a standard intervention involving 1008 children aged 1–17years of age presenting with acute asthma to one of the five participating Canadian paediatric EDs. The study includes a nested cohort study of children who presented at selected centres and who could cooperate with eNO measurements (about 200) or induced sputum (about 60 children aged ≥8 years) to explore the pattern of airway inflammation.

Subjects

Participants will be eligible if they: (1) are aged 1–17 years; (2) have asthma, defined as: (i) prior diagnosis of asthma made by a physician; (ii) prior documented episode of acute cough, wheezing and/or dyspnoea with significant response to inhaled β2-agonists or to oral cor- ticosteroids; (iii) in a child aged3 at baseline; (5) have a good understanding of English or French and (6) are accom- panied by parents or legal guardians.

Patients will be excluded if they present: (1) with another chronic respiratory condition (such as broncho- pulmonary dysplasia or cystic fibrosis); (2) with a reason- able suspicion of bronchiolitis or foreign body aspiration; (3) a history of hypersensitivity to salbutamol, ipratropium bromide or oral prednisolone; (4) a relative or absolute contraindication to receiving oral corticosteroids such as recent exposure to varicella or live vaccine in a susceptible child or (5) with confirmed or sus- pected pregnancy status. As some inclusion and exclu- sion criteria may become evident during or after the ED stay (ie, pneumonia), a central adjudicating committee comprising a paediatrician/ED physician and respirolo- gist will review the eligibility criteria of children who, after enrolment, appear to be ineligible due to the failure to meet one or more inclusion or exclusion cri- teria and who did not receive or tolerate oral corticoster- oids as recommended in the protocol. The main trigger for identifying these children will be the ED physician’s low degree of confidence that this child had asthma, sus- picion that he/she may have bronchiolitis and the suspi- cion that he/she did not retain the oral corticosteroids. In this case, the relevant case report forms, relevant anonymised laboratory testing (ie, imaging, microbiol- ogy, virology and haematology) and relevant anonymised medical chart sections will be reviewed by the adjudicat- ing committee. The adjudicating committee will be central, although we are not ruling out the possibility of creating local committees at each site, depending on the volume of cases to review. In such a case, the site would be added as a covariate in the analysis. Disagreement will be resolved by consensus or the assistance of a third observer. The proportion of excluded children, assumed to be around 10%, will be monitored and reported.

Standardised acute treatment protocol

As per the evidence-based paediatric national and international guidelines, children will be treated according to a standardised severity-specific protocol (table 1). All children will receive 2 mg/kg (maximum 50mg) of oral prednisolone (or prednisone) within 60 min of triage, along with inhaled salbutamol with or without inhaled ipratropium bromide as per severity strata. Discharge medications will include a 5-day course of oral prednisolone at a dose of 1 mg/kg/day and inhaled β2-agonists as needed. Cointerventions, such as add-on therapy with magnesium sulfate in the ED or inhaled corticosteroids at discharge, will be permitted and recorded. Comorbidities (eg, pneumonia, sinusitis, allergic rhinitis, etc) and adverse health events will be documented.

Measurements of effectiveness

The primary outcome, serving as a proxy for suboptimal response, is the failure of ED management defined as a hospital admission for asthma (as primary or secondary diagnosis if the primary diagnosis is a complication or comorbidity of asthma) or ED stay for asthma (with active treatment) of ≥8 h after intake of oral corticosteroids or a return visit within 72 hours meeting either of the two previous criteria (admission or length of active treatment ≥8 hours). The 8 h time constraint was included to account for extraneous factors affecting admissions, such as variation in bed availabilities while the child remains under treatment on site.

Secondary measures of effectiveness in the ED include: (1) meeting the severity criteria for admission, that is, a PRAM score ≥4 within 4 h of corticosteroid administra- tion (to account for other reasons for hospital admission); (2) the PRAM profile in the ED, measured hourly from baseline and every hour until disposition (or until 4 h after intake of oral corticosteroids, whichever occurs first) and reported as the area under the curve; (3) time to PRAM ≤3, that is, meeting the criteria for dis- charge; (4) length of active treatment defined as duration between the first and last salbutamol inhalation. Finally, to allow more objective and precise quantification of response in a nested cohort (CHU Ste-Justine), (5) change in Rrs between baseline and disposition will be documented on the MasterScreen Impulse Oscillometry (Cardinal Health Canada, Montreal, Canada) using pre- viously described standardised techniques in cooperative children aged ≥3 years, (6) change in eNO between baseline and disposition will be documented on a chemiluminescence NO analyser in cooperative chil- dren aged ≥4years and (7) hospital admission for asthma (as primary or secondary diagnosis) within 72 h or an active ED stay of ≥8h after intake of oral corticosteroids.

Secondary measures of resolution of exacerbation mea- sured after discharge will be documented over the next 10 days in all patients: (8) unscheduled visits for asthma as reported by parents and confirmed by medical charts; (9) cumulative symptom score and duration of symptoms measured daily on the validated Asthma flare-up diary for young children (formerly named the Paediatric Asthma Diary); (10) parent quality of life measured on the ‘Effect of a child’s asthma flare-up on parents’ and (11) the cumulative number of puffs and duration of use of rescue β2-agonists as recorded on the Asthma flare-up diary for young children.

Potential determinants

Sociodemographic variables

Basic characteristics will be documented, including age, gender, neighbourhood financial income derived from six-digit postal codes from both parents (if separated), medical insurance (private or public) and ethnicity as reported by the parents and classified as per the latest Canadian Census questionnaire.

Phenotype and morbidity

Children’s asthma phenotype will be documented as per the latest international recommendations (ie, viral induced, exercise induced, allergen induced and mul- tiple trigger). Prior morbidity (admissions, ED visits and rescue oral corticosteroids), asthma control using the Asthma Quiz for Kidz, reported use of daily con- troller medications, environmental factors (eg, dust, animals and pollen) and perceived trigger of the exacer- bation will be documented. Prior allergy test results, serum IgE (specific) will be obtained (with permission) as well as pollen counts in the area.

Viral trigger

Acknowledging viruses as the major trigger, all children will be tested for respiratory viruses. All children will have a nasopharyngeal aspirate or nasopharyngeal swab (Flocked swab, Copan Diagnostics, California, USA) per- formed. The aspirate or swab will be put in 3 mL of viral transport media (UTM, Copan Diagnostics, California, USA) and split in half on site. The first half will be tested using routine methods in each site if clinically required and the other half will be frozen at −80°C for molecular diagnosis. The frozen samples will be pro- cessed using the validated automated microarray detec- tion, which tests for 23 common respiratory viruses, including the novel influenza A/H1N1/Mexico.

Passive exposure to tobacco smoke will be quantified by a questionnaire specifically enquiring about current smoking and the amount of smoking by household members, cumulative smoke exposure in pack-years since birth and in utero exposure, using validated questions. Active smoking will be assessed by asking school-aged children aged 10 years and older, while alone, if they smoked in the past 7 days, using standardised questions used by the Quebec Institute of Statistics. A quantitative cotinine by quantitative enzyme immunoassay kit (Salimetrics, Pennsylvania, USA) will be performed on saliva sampled using three Sorbettes (a wand with a small sponge) accord- ing to a previously described protocol.

Several other factors will be considered, including an assumed trigger. In the absence of valid biomarkers of causal relationship to the exacerbation, the alleged trigger (allergic or other) based on the parental report, physician perception, prior documented sensitisation (skin tests or specific IgE), pollen, particles, ozone (and other) levels in the child’s living area will serve to infer a trigger.

Mechanistic pathway

Genetic profile

Patients’ DNA will be extracted from saliva expectoration or collected on Sorbettes designed for young children, with only a small amount (1–2 mL) needed for DNA analysis. After amplification by PCR, key polymorph- isms will be determined by high throughput geno- typing technology (Sequenom platform) for custom SNP panels.

eNO will be measured on a chemiluminescence ana- lyser (the Niox Flex or the portable Niox Mino from Aerocrine, New Providence, New Jersey, USA or Sievers from GE Analytic Instrument, Boulder, Colorado, USA), using standardised techniques in cooperative children aged ≥4 years; the measurements will be taken before corticosteroids and at 4h or disposition, whichever occurs first. In children unable to cooperate with these techniques, the eNO will also be measured, using the single breath manoeuvre, in an inert balloon (offline technique) and then analysed on the chemilumines- cence analyser.

Induced sputum

After the initial salbutamol inhalation, children aged ≥7 years will be asked to expectorate spontaneously or, if unsuccessful, receive a nebulisation of 0.9% saline solu- tion over 7–10 min at 5 L/min with oxygen. With this technique, a 70% success rate was obtained among acutely ill school-aged asthmatic children. Throughout the induction, lung function tests using Rrs will be documented to ensure that the coughing effort does not induce bronchospasm. Sputum will be pro- cessed according to standard techniques within 8h. Briefly, sputum will be separated from the saliva and dis- persed using dithiothreitol. The dispersed suspension is then centrifuged and filtered; cytospin slides will be pre- pared on site by a trained medical technologist, and the supernatant frozen at −70°C for subsequent quantifica- tion of inflammatory mediators. Differential cell counts and inflammatory mediators will each be analysed by an independent laboratory, blinded to exposure and outcome.

Procedures

Within each institution, potentially eligible patients aged 1–17 years will be identified on arrival in the ED. As per standard practice in each of the participating hospitals, all children will be triaged and scored on the PRAM at or shortly after triage. The first inhalation of salbutamol and, for severe exacerbations, ipratropium bromide will be administered (figure 1). Using a two-step informed consent, parents will first give their authorisation for their child to receive standardised severity-specific treat- ment and, at the CHU Ste-Justine, lung function testing and induced sputum in cooperative children. Parents will then receive a detailed explanation of the study and be offered study participation. Participants will receive the treatment and measures detailed in figure 1, with key measurements obtained prior to corticosteroids.

Statistical analyses

Standard summary statistics (N, mean, SD, median, minimum and maximum for continuous variables; N and proportion for categorical variables) will be computed for all variables for the whole cohort and by institutions. Two-sided 95% CIs will be presented as necessary. For the primary endpoint, failure of ED management and other dichotomous outcomes, bivariate and multivariate logis- tic regression models will be used to examine the associ- ation with each potential determinant and to adjust for site, baseline severity and other relevant covariates of interest. ORs will be estimated and presented with two- sided 95% CIs. Continuous outcomes, such as the area under the curve for repeated PRAM measures, daily Asthma flare-up diary for young children or intensity of both β2-agonist use and symptom scores after discharge, will be analysed using linear regression models. Transformation of variables will be performed if neces- sary to account for non-normality of the residuals.

With regard to pharmacogenomics, we will compare the frequency of SNPs between patients with versus without failure of ED management using Fisher’s exact tests and examine the strength of the association with failure of ED management with bivariate and multivariate logistic regression. Genotypes will be considered as vari- ables with two (dominant or recessive models) or three (additive model) categories. The choice of the model is determined by the functionality of selected polymorph- isms and previous reports. Bonferroni correction will be used to adjust for multiple testing (p=0.005, adjusted for 10 independent genes investigated). Host–environment interaction will be examined by adding other covariates of interest to the genotype model. For example, to investi- gate genetic heterogeneity according to early-life expos- ure to ETS, we will introduce markers of tobacco smoke exposure status.

With regard to examining eosinophilic inflammation as a marker of responsiveness, the area of the curve of serial PRAM measurements and time to PRAM <4 will serve as outcomes. Linear regression will be used to examine the bivariate and multivariate relationships between sputum eosinophils counts, patterns of inflam- mation (eosinophilic, non-eosinophilic and mixed) and baseline eNO with the PRAM profile (area under the curve); the standard survival curve will explore the dif- ference in time to meet criteria for discharge, censored at 4 h. All models will be checked using appropriate regression diagnostics. Results will be reported as signifi- cant when p<0.05.

Sample size

Sample size was estimated in two steps using hospital admission as the dependent variable in a multivariate logistic regression model. Pilot data were extracted from two large recent chart audits totalling 1628 chil- dren presenting with acute asthma to the ED (in which age, gender, baseline PRAM, timing of oral corticoster- oid intake and admission were documented) and two recently completed trials totalling 518 children, in whom tobacco exposure was also ascertained by ques- tionnaire; we then focused only on children meeting our current eligibility criteria (age, baseline PRAM and corticosteroids within an hour of arrival). Based on the literature, we assumed the prevalence of viral pathogen varied between 60% and 80%. The baseline risk of admission was 41%; after recruitment of the initial 320 patients, the observed overall risk of admission was sub- stantially lower at 16%, which substantially reduced the effective sample size from 1200 to 800. First, using the Wald test (based on the prevalence of factors and the risk of admission in unexposed children), we calculated the sample size required to detect an RD of admission of 7.5%, taking each determinant in turn as the inde- pendent variable in a bivariate logistic regression; we used 95% CIs for each estimate obtained from pilot data to examine the impact on power. Then, using the method proposed by Hsieh et al, we calculated the inflation factor to adjust the total sample size, based on the observed correlation between key determinants age, gender and tobacco exposure, and baseline sever- ity). As the observed correlation between determinants and severity and any pair of determinants was negligible (β coefficients

Figure 1 The treatment protocol is stratified on the severity of asthma exacerbation, that is, moderate or severe, as measured by the validated Pediatric Respiratory Assessment Measure (PRAM). Categories of medications are listed in the three columns. The three horizontal panels describe therapy administered in the first 60 minutes (top panel); after the initial 60 minutes (middle panel); and on discharge (bottom panel).

ETHICS AND DISSEMINATION

This study poses little risk to participants, as all children will receive state-of-the-art asthma management with oral corticosteroids, an approach associated with minimal side effects. Children will be assessed for potential contraindication to corticosteroids prior to enrolment. Using a two-step informed consent, parents will first give their authorisation for their child to receive the severity- specific asthma protocol and, at specific centres, lung function testing and induced sputum in cooperative chil- dren; then they will consent to the full study including sampling for cotinine, DNA and viruses. All parents will sign an informed consent form authorising the DNA ana- lysis for the specified research purpose as well as future analyses (including biobanking of DNA in anonymised fashion), provided these are approved by the institutional ethics boards. Assent will be obtained from children aged ≥7 years. The collection of saliva by expectoration or sponge is painless and well tolerated by children. Nasopharyngeal aspiration or swab for viral diagnosis is a commonly used procedure in the ED setting; it is rapid and associated with little discomfort. The performance of Rrs and eNO is non-invasive and devoid of serious side effects. Sputum induction with 0.9% saline and salbuta- mol pretreatment is a safe procedure in the acute care setting. Data safety is maximised through: restricted physical access to computers and electronically protected servers (firewall, restricted password-protected

Downloaded from bmjopen.bmj.com on April 7, 2014 - Published by group.bmj.com access, daily back-up and storage in 2 different locations). Confidentiality is also a part of standard operating proce- dures; a unique research code will be assigned to each patient. All research data and specimens will be kept in locked rooms with restricted access at each site, the coordination centre and the site of analysis. The proto- col, consent form and any amendments are subject to the ethics review board approval of each institution. Each investigator will adhere to the principles of the Declaration of Helsinki. All serious adverse effects will be promptly reported.

This study will provide needed answers as to whether oral corticosteroids are less effective for preventing hos- pital admission in subgroups of children and, if so, which clinically available characteristics could be used to identify patients at risk of poorer response. An impaired response to systemic steroids in certain subgroups, resulting in a higher rate of failure of ED management and/or impaired recovery, would challenge the current standard of practice and call for an immediate search for better approaches. Given the high prevalence of suspected factors (young age (60%), viral infection (60–80%), ETS (20–30%)), the potential impact of poor response on the rate of failure of ED manage- ment is sizeable. The exploration of potential host– environment interactions will broaden our understand- ing of corticosteroid responsiveness in children and enable us to focus on relevant alternative/supplemen- tal strategies to adequately manage these patients. Documentation of the similar effectiveness of systemic corticosteroids across determinants will provide the needed reassurance as to the value of national recom- mendations for the treatment of all children with acute asthma.

Results will be disseminated at international confer- ences targeting emergency physicians, paediatricians, geneticists and respirologists. Four manuscripts are envi- sioned: one pertaining to the identification of main determinants of the risk of failure of ED management within 72 h of the dose of oral corticosteroids (general medical journal); one pertaining to the validation of perceived triggers and clinical predictors of response to therapy during the index exacerbation (emergency medicine journal); one pertaining to the genetic poly- morphisms associated with non-response to oral corticos- teroids (respirology journal); and one pertaining to the gene–environment interaction modulating the response to corticosteroids in children (paediatric journal).